Cervical cancer diagnostics – the challenge

The introduction of Pap testing was followed by an enormous decrease regarding the incidence of cervical cancer. Since this development is stagnant for at least a decade, more sophisticated screening tools are needed. Therefore testing for high-risk types of human papillomaviruses (HPV) has become an important part of cervical cancer (CxCa) screening, management and treatment. Although highly sensitive, hrHPV testing has only limited specificity. Consequently, triage methods following a positive hrHPV test result are needed to avoid unnecessary follow-up of women who are infected without having clinically relevant disease.

The new PCR-based assay GynTect® can be used as a triage test for HPV-positive women, allowing to identify those with cervical (pre)cancer.

The rationale for GynTect®

Persistent infection with a high-risk HPV type leads to genetic instability of infected cervical cells due to the expression of active viral oncogenes E6 and E7. Consequently, different genomic changes occur in the course of a normal cell developing into a cancer cell. Among these changes are epigenetic modifications involving the hypermethylation of specific genes, also called DNA methylation.

GynTect® detects DNA methylation of six human marker gene regions which specifically arises during car-cinogenesis. As these genes are reliably found methylated in cervical (pre)cancer cells, GynTect® detects malignant host cell transformation instead of viral infection.

GynTect®Epigenetic biomarkers for reliable

cervical cancer diagnostics

Clinical Performance of GynTect®

The clinical performance of GynTect® was evaluated in a clinical trial using 94 cervical scrapes collected for routine HPV testing in the colposcopy clinic Jena. All samples were tested positive for hrHPV and his-topathological results were available for all samples. 52 patients were ≥ 30 years of age, 42 < 30 years of age. 55% of all samples had a negative histopathological finding (no CIN); in 21 cases CIN3 and in 4 cases cervical carcinoma (CxCa) was diagnosed (see Table 1).

GynTect® detected all cervical carcinoma. Only 4% of the samples with negative histopathological find-ing were GynTect®-positive in the older age group, which could significantly reduce unnecessary colpos-copies (see Table 2). The clinical performance is depicted in Table 3. GynTect® showed high sensitivity and specificity for the detection of CIN3+ in HPV-positive women 30 years and older. In younger women (<30 years of age), GynTect® performed less sensitive regarding the detection of CIN3. But especially in younger women many CIN2 and CIN3 can regress spontaneously1,2. Therefore, future work might provide more information that a certain proportion of CIN3 cases in younger women might not need to be detect-ed yet.

Based on the current data, GynTect® is suitable for triaging HPV positively tested women older than 30 years of age. Besides, it is intended as an aid in the diagnosis of cervical (pre)cancer.

Table 1: Study cohort stratified for histopathology finding & age groups

Table 3: Clinical performance (CIN3+) stratified for age groups.

Table 2: Detection rates stratified for histopatho-logy finding & age groups

1. Trimble et al. 2005 Spontaneous regression of high-grade cervical dysplasia: effects of human papillomavirus type and

HLA phenotype. Clin Cancer Res. 2005 Jul 1;11(13):4717-23.2. Ostör 1993 Natural history of cervical intraepithelial neoplasia: a critical review. Int J Gynecol Pathol. 1993 Apr;12(2):186-92.

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women ≥ 30 years

women < 30 years

total

no CIN 27 25 52

CIN1-2 10 7 17

CIN3 11 10 21

carcinoma 4 0 4

women ≥ 30 years

women < 30 years

no CIN 4 % 0 %

CIN1-2 30 % 15 %

CIN3 72 % 20 %

carcinoma 100 % —

women ≥ 30 years

women < 30 years

Sensitivity 80 % 20 %

Specificity 89 % 97 %

Step 1: Gynecologist visit

Abnormal Pap smear (Pap-III, Pap-IIID)? And positive HPV test result? Your patient is uncertain and wants further clarifi cation? GynTect® off ers you as a gynecologist the option to further clarify the status of your patient: GynTect® allows to distinguish be-tween cervical lesions that require invasive diagnostics and eventually surgery, and those that may heal without treatment.

Step 2: Cervical sampler

GynTect® is performed using conventional cervical smear material, which is trans-ferred to a specifi c transport medium. Currently the smear material is transferred to STM medium, which QIAGEN has developed for the HC2 HPV test. Please contact your diagnostic lab or oncgnostics via the website, by email, fax or phone in order to receive a cervical sampler.

Step 3: Sampling and delivery to laboratory

Use the cervical brush from the sampler and take a smear, transfer the smear material to the transport medium. The sample may be sent by normal mail, and should arrive at the diagnostic lab or at oncgnostics within 48h. The cost for GynTect® is invoiced di-rectly to the patient, who will either send it to her health insurance or pay it by herself.

Step 4: Performance of GynTect®

The laboratory performs GynTect® within a work day. The sample is prepared in the laboratory, and the GynTect® markers are detected by the PCR-based test. You re-ceive a result within a few days.

Step 5: Clear decision

You discuss the results with your patient. In case of a negative result your patient should not have any severe lesion that may proceed to carcinoma. You may observe your patient following a watchful waiting strategy, with repetition of the HPV test in order to determine when the infection clears.

In case of a positive GynTect® result a colposcopy and biopsy should provide clari-ty, where at the cervix the disease is located. This allows a specifi cally-targeted intervention.

GynTect® – assay principle and workfl ow

The GynTect® assay principle is based on the detection of DNA methylation of human marker gene regions. DNA methylation is a process during which methyl groups are added to the DNA, more specifi cally, to cyto-sines followed by guanines, so-called CpG dinucleotides.

The analysis of a patient sample comprises two steps. First, the methylation status of the human DNA in a cervical sample is “fi xed” by a so-called bisulfi te treatment. In the second step the bisulfi te-converted DNA is analyzed in the regions of interest by applying several sensitive real-time PCR reactions. Using spe-cifi c PCR primers, only the originally methylated DNA marker regions are selectively amplifi ed, and a PCR product can be detected. Therefore, this procedure is called methylation-specifi c PCR (MSP). The GynTect® assay is rated positive, if a defi ned set of markers is amplifi ed.

For a highly reliable workfl ow, the assay includes several sample quality control reactions. Additionally, sep-arate positive and negative control materials are provided with the kit.

Benefi ts of GynTect®

As a triage test based on cervical cancer cell biomarkers, GynTect® was developed to identify those indi-viduals among HPV positively tested women who have clinically relevant cervical disease. Consequently, one may recommend colposcopy-guided diagnostics if the test is positive. A negative test results indicates the absence of a severe lesion or carcinoma and allows the continuation of a non-invasive screening algo-rithm, certainly with a shorter screening interval due to the initial positive HPV test result. Generally, DNA methylation-based biomarker tests may be applied in diff erent cervical cancer screening settings, e.g. as triage in an HPV test-based screening scenario or for the clarifi cation of minor cytological cervical lesions. GynTect® is easily incorporated in current gynecologists’ practices as it may be performed using residual material from e.g. the HPV test.

Patient sample:direct cell lysis or

DNA isolation

1 2 3 4

Hands-on time: 5-10 minSample Collection via

STMTM (Qiagen). Coming soon: Preserv-

CytTM (Hologic)

Positive GynTect test result

» Indicates the presence of a clinically relevant cervical disease

» colposcopy-guided diagnostics with biopsy sampling for histopathology, if necessary

GynTect: advantages for the gynecologist

» objective test result can be provided to the patient

» visit of a specialist is not needed in any case

» next steps can be recommended directly, watchful waiting can be avoided

» no change in the current practice

Negative GynTect test result

» Indicates the absence of a severe lesion or carcinoma

» continuation of a non-invasive screening algorithm with shorter interval due to the initial positive HPV test

GynTect: advantages for the patient

» no long-term uncertainties between fi rst diagnosis and clarifi cation

» avoid unnecessary invasive diagnostics and surgeries

» classify an HPV-positive test result

» no change in the current practice

Bisulfi te treatment: “Fixation” of

DNA methylation

Duration: 90 minThereof hands-on time

40 min. Bisulfi t conversion is

done with the EpiTect® Fast Bisulfi te Kit

(Qiagen). Order via oncgnostics possible.

Analytical PCR: detection of the

DNA marker regions

Duration: 130 minThereof hands-on time

5-10 min. GynTect® runs on the ABI7500 or 7300 real

time PCR system (Life technologies).

More platforms to come.

Data analysis:Evaluation of the PCR

result

Hands-on time: 30 minThe algorithm if

GynTect® is scored positive is applied using a calculation software like Microsoft Excel.

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